AU2017221883A1

AU2017221883A1 - Compounds, pharmaceutical compositions and a method for the prophylaxis and treatment of the adhesion process

Info

Publication number: AU2017221883A1
Application number: AU2017221883A
Authority: AU
Inventors: Mikhail Gennadievich SHURYGIN; Irina Aleksandrovna SHURYGINA
Original assignee: Pharmasyntez JSC
Current assignee: Pharmasyntez JSC
Priority date: 2011-05-17
Filing date: 2017-09-01
Publication date: 2017-09-28
Anticipated expiration: 2032-05-17
Also published as: AU2012257347A1; JP2016175931A; CL2013003305A1; CR20130600A; CN103534293B; IL229423B; MX2013013409A; RU2013155582A; HK1189012A1; BR112013029616B1; JP5936683B2; US10493164B2; LT2716684T; CO6801779A2; KR20170034950A; CA2836268C; AP3694A; RS59382B1; JP2014515057A; SV2013004582A

Abstract

Abstract The present invention relates to the fields of pharmacy, clinical and experimental medicine and veterinary science. In particular, it relates to new compounds showing inhibiting effects on p38 MAP-kinase structures of types (I) (VII) that may be used for the treatment and prevention of adhesions. The invention describes pharmaceutical compositions containing a sufficient amount of SB203580 or any of type (I)-(VII) compounds, or their combinations and a pharmaceutically acceptable carrier, diluent or excipient. Provided is a pharmaceutical composition SB203580 used as a compound showing an anti adhesion activity. In addition, this invention provides methods for the prevention and/or treatment of diseases or conditions that serve as risk factors for adhesion formation and/or development allowing physicians to avoid additional administration of drugs in the postoperative period.

Description

Compounds, pharmaceutical compositions and a method for the prophylaxis and treatment of the adhesion process

FIELD OF INVENTION

The present invention relates to the fields of pharmacy, clinical and experimental medicine and veterinary science. In particular, it relates to new compounds for the treatment and prevention of adhesions, pharmaceutical compositions containing these compounds, and to a method for the treatment and prevention of adhesion formation. Compounds described in this invention show inhibiting effects on p38 MAP-kinase.

BACKGROUND OF THE INVENTION

Adhesion formation is a topical problem of clinical medicine. Since adhesion especially often inhibits normal movement of tissues, including organs, it is considered as a serious complication after surgery. The incidence of intraperitoneal adhesions ranges from 67 to 93% after general surgical abdominal operations and up to 97% after open gynaecological pelvic procedures.

It has been estimated that in the United States, there are 117 hospitalisations for adhesion-related problems per 100,000 people, and the total cost for hospital and surgical expenditure is about $1.3 billion [Abdominal adhesiolysis: inpatient care and expenditures in the United States in 1994/ N.F. Ray, W.G. Denton, M. Thamer et al.//J. Am. Coll. Surg.- 1998.- Vol. 186.- P. 1-9].

The main approaches in preventing adhesions include adjusting surgical techniques, limiting trauma to intra-abdominal structures, and applying adjuvants to decrease adhesion formation [Risberg B.O. Adhesions: Preventive strategies/ B.O. Risberg// Eur. J. Surg. Suppl.- 1997.- Vol. 577.- P. 32-39],

However, the use of some medicinal preparations for the prevention of adhesions is limited by the following factors: 1) Ischemic zones are at risk of adhesion formation, but they are distant from blood flow and thus from pharmacological effects of medications administered by common routes (per os, intravenously, intramuscularly, etc.); 2) An extremely rapid absorption mechanism which is typical for the peritoneal membrane affects the elimination half-life and efficacy of many medicinal agents administered intraperitoneally; 3) Any anti-adhesion agent should show its specific activity against the adhesion formation but not the normal wound repair which is necessary for adequate surgical treatment.

Intra-peritoneal thrombokinase, fibrinolysin, streptokinase, urokinase, hyaluronidase, chymotrypsin, trypsin, papain, and pepsin act directly by breakdown of the fibrinous mass and indirectly by stimulating plasminogen activator activity. The use of these agents is still awaiting appropriate human clinical trials [Alpay Z. Postoperative adhesions: from formation to prevention/ Z. Alpay, G.M. Saed, M.P. Diamond// Semin. Reprod. Med.- 2008.- Vol. 26, N 4.- P. 313-321],

The use of non-steroidal anti-inflammatory agents, glucocorticosteroids and antihistamines, progesterone/estrogen, anticoagulants, fibrinolytics, and antibiotics has not been found very effective in reducing adhesions and has been associated with an inadequate safety profile and a high incidence of various side effects [Pathogenesis, consequences, and control of peritoneal adhesions in gynaecologic surgery/ Practice committee of the American society for reproductive medicine, The society of reproductive surgeons// Fertil. Steril.- 2008.- Vol. 90, Suppl. 5.- S. 144-149], A pathologically justified approach to the prevention of adhesions is the use of methods and agents preventing approximation and adhesion of injured abdominal surfaces [Davey A.K. Surgical adhesions: A timely update, a great challenge for the future/ A.K. Davey, P.J. Maher// J. of Minimally Invasive Gynecology.- 2007.- Vol. 14.-P. 15-22],

An ideal barrier showing a high safety and efficacy profile should be noninflammatory, nonimmunogenic, and persist during the critical remesothelisation phase, staying in place without sutures or staples, and furthermore remain active in the presence of blood and be completely degradable. In addition, it should neither interfere with healing, promote infection and oncological process, nor itself cause adhesions [Yeo Y. Polymers in the prevention of peritoneal adhesions/ Y. Yeo, D.S. Kohane// European. J. of Pharmaceutics and Biopharmaceutics.- 2008.- Vol. 68.- P. 57-66],

Nowadays, polymer solutions [Falabella C.A. Cross-linked hyaluronic acid films to reduce intra-abdominal postsurgical adhesions in an experimental model/ C.A. Falabella, W. Chen// Dig. Surg.-. 2009.- Vol. 26, N 6.- P. 476-481], solid membranes [Hyaluronan derivatives in postsurgical adhesion prevention/ D. Pressato, E. Bigon, M. Dona et al. // in: Hyaluronan: Proceedings of an International Meeting, September 2000, North East Wales Institute, UK, Woodhead Publishing, Cambridge, England, 2002. - P. 491-499], precasted [A novel hyaluronan-based gel in laparoscopic adhesion prevention: preclinical evaluation in an animal model/ P.A.D. Laco, M. Stefanetti, D. Pressato et al.// Fertil. Steril.- 1998.- Vol. 69.- P. 318-323] or in situ hydrogels [Next-generation hydrogel films as tissue sealants and adhesion barriers/ S.L. Bennett, D.A. Melanson, D.F. Torchiana et al.// J. Card. Surg.- 2003.- Vol. 18.- P. 494-499] are used as these barriers preventing adhesion formation.

The use of crystalloid solutions for long-term separation of abdominal layers has been found inappropriate due to the rapid absorption of water and electrolytes from peritoneal cavity, with up to 500 ml of iso-osmolar sodium chloride absorbed in less than 24 hours in humans [Kinetics of peritoneal fluid absorption in adult man/ L. Shear, C. Swartz, J. Shinaberger et al.// N. Engl. J. Med.- 1965.- Vol. 272.- P. 123-127], Because it takes 5 to 8 days for peritoneal surfaces to remesothelialise, a crystalloid solution should be absorbed well before the process of fibrin deposition and adhesion formation are complete. Clinical studies showed an adhesion re-formation rate of approximately 80% in patients who received crystalloid solutions [De Chemey A.H. Clinical problem of intraperitoneal postsurgical adhesion formation following general surgery and the use of adhesion prevention barriers/ A.H. De Chemey, G.S di Zerega// Surg. Clin. North. Am.-1997.- Vol. 77.-P. 671-688],

Attempts have been made to use different polymer materials, in particular polymers of glucose (Dextran 70, isodextrin), carboxymethyl cellulose, and hyaluronic acid.

Dextran 70 (32% dextran 70 (Hyskon, Pharmacia, Sweden)) is a frequently used solution for adhesion prevention. Its main characteristics are as follows: dextran is slowly absorbed and draws fluid into the abdominal cavity. It also decreases clot formation [Gutmann J.N. Principles of laparoscopic microsurgery and adhesion prevention/ J.N. Gutmann, M.P. Diamond// in: Practical Manual of Operative Laparoscopy and Hysteroscopy: Ed. Azziz R., Murphy A.A.- New York: Springer, 1992.- P. 55-64], Follow-up studies of the initial observation did not show a reduction in adhesions. Moreover, significant side effects, such as ascites, weight gain, pleural effusion, labial edema, liver function abnormalities, and, albeit rare, disseminated intravascular coagulation and anaphylaxis, were noted, and dextran solution is used very rarely now. [di Zerega G.S. Contemporary adhesion prevention/ G.S. di Zerega// Fertil. Steril. - 1994.- Vol. 61.- P. 219-235], The results have been inconsistent [Tulandi T. Intraperitoneal instillates/ T. Tulandi// Infertil. Reprod. Med. Clin. North. Am.- 1994.- Vol. 5.- P. 479-483]

Difficulties have been found in using porous polytetrafluoroethylene membranes as local barriers due to the formation of pseudocapsules [The Surgical Membrane Study Group: Prophylaxis of pelvic sidewall adhesions with Gore-Tex surgical membrane: A multicentre clinical investigation// Fertil. Steril.- 1992.- Vol. 57.- P. 921-923], Moreover, it has been technically difficult to use this material in laparoscopic surgery [Tulandi T. Adhesion prevention in laparoscopic surgery/ T. Tulandi// Int. J. Fertil. Menopausal. Stud.- 1996.- Vol. 41.- P. 452-457], It requires a physical fixation and is not degradable. Therefore, it should be left or surgically removed later. Removal procedures may cause surgical traumas and lead to adhesion formation. These technical difficulties and usability problems have made the medication unpopular, and it is used very rarely now.

Oxidised regenerated cellulose (Interceed) is the only adjuvant approved for the specific purposes of postsurgical adhesion prevention. ORC appears to decrease adhesion formation-reformation beyond that achieved with meticulous surgical technique. ORC reduces both raw surface area and the occurrence of adhesion formation-reformation by a margin of 20% [Interceed (TC7) Adhesions Barrier Study Group: Prevention of postsurgical adhesions by Interceed (TC7), an absorbable adhesion barrier: A prospective, randomized multicenter clinical study//Fertil. Steril.- 1989.- Vol.51.- P. 933-938], When applied to a raw peritoneal surface, it becomes gel within 8 hours [Synergistic effects of Interceed (TC7) and heparin in reducing adhesion formation in the rabbit uterine horn model/M.P. Diamond, C.B. Linsky, T. Cunningham et al.//Fertil. Steril.- 1991.- Vol.55.- P. 389-394], ORC can be applied easily by laparoscopy, and does not need suturing. However, clinical observation indicates that small amounts of bleeding at the time that ORC is applied results in blood permeating the weave of the material. Fibroblasts grow along the strands of clotted blood with subsequent collagen deposition and vascular proliferation [Frankfurter D. Pelvic adhesive disease/D. Frankfurter, A.H. De Chemey//Postgrade Obstet. Gynecol.- 1996.- Vol. 16.- P. 15], This means that the presence of intraperitoneal blood negates any beneficial effect [Effect of blood on the efficacy of barrier adhesion reduction in the rabbit uterine horn model/C.B. Linsky, M.P Diamond., G.S. di Zerega et al.//Infertility.-1988.-Vol.l 1.-P. 273-280],

Summing up the current approaches to the prevention of postoperative adhesions in the peritoneal cavity, Burlev V.A. et al. (2009) [Burlev V.A. Peritoneal adhesions: pathogenesis and prophylaxis. V.A. Burlev, E.D. Dubinskaya, A.S. Gasparov//Reproductive disorders.- 2009.- No.3.- P. 36-44] regret to state that all current methods for adhesion prevention are insufficiently effective (and quite expensive as well) and further studies are required to improve the efficacy of anti-adhesion measures.

The most similar to the present invention in technical terms is a method for the prevention of adhesions consisting in the injection of a combination of sterile Lintex-Mesogel gel and derinate into the serous sac [Method for the prevention of postoperative adhesions: Patent 2363476 of the Russian Federation: ΜΚΠ51: A61K31/711, A61K31/717, A61P41/00 / Gomon M.S., Lipatov V.A., Konoplya A.I., Bezhin A.I., Loktionov A.L., Kasyanova M.A., Sukovatykh B.S., Godova A.Yu.; patent applicant/holder Gomon M.S., Lipatov V.A.- No. 2007147670/14; submitted on December 20, 2007; published on August 10, 2009, Newsletter No. 22.- 6 pages].

This method for the prevention of adhesions consists in the following. During abdominal operation, for example, laparotomy or laparoscopy, and/or before the covering of the serous sac at the final stage of the surgical intervention, the areas with high probability of primary or recurrent adhesion development (for example, deseronised areas, anastomotic areas, areas with acute or possible inflammation, trauma zones after adhesion dissection, areas of abdominal drying, etc.) are treated with sterile Lintex-Mesogel gel and depot derinate. The volume of derinate is 1% to 25% of total mixture volume. The combination of the derinate and polymer gel is achieved when a mixture is prepared extemporaneously immediately before use, with the correct proportions of components. The ratio of gel-to-derinate solution volumes (based on 1.5 mg of derinate in 1 kg) should be such as the injected solution does not exceed 25% of the total volume, because more fluid may reduce the viscosity of the gel and its anti-adhesion activity. For adhesion prevention purposes, a portion of gel is applied to the serous surface using a syringe or squeezed into the palm of the surgeon’s hand from the container where the mixture has been prepared, and applied by smooth movements on the raw abdominal surface, deseronised areas and areas where adhesions may occur (areas with signs of inflammation or ischemia: edema, hyperemia, dilated vessels, discoloration, peristalsis, decreased pulsation of abdominal vessels, etc.). When diffusion processes occur (for example, after abdominal sanation in patients with generalised peritonitis), the combination of gel and derinate is applied at a dose calculated according to the table mentioned by G. DiZerega (1999 r.), that is 2.4 ml/kg for humans, and 10.7 ml/kg for animals (rats). When laparoscopic procedures are performed, specific injectors are used to apply gel with depot derinate.

Disadvantages of this method include the necessity to prepare the sterile solution during the operation, which can make the surgical process more complicated. Other disadvantages include difficulties in achieving homogeneity, difficulties in derinate dosing (need to be weighed), the need to use specific injectors/manipulators in laparoscopic procedures, and an absence of components inhibiting the activity of fibroblasts - cells which synthetic activity stimulates adhesion formation.

The p38 MAP-kinase inhibitor SB203580 is known to be an inhibitor of proinflammatory cytokine production [Badger A.M., Bradbeer J.N., Votta B. et al. Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function// J. Pharmacol. Exp. Ther.-1996.- Vol. 279.- P. 1453 - 1461],

However, no data were obtained by the inventors and no literature references were found concerning the use of p38 MAP-kinase inhibitor as an agent exhibiting anti-adhesion activity.

DETAILED DESCRIPTION OF THE INVENTION

The purpose of the present invention is to develop compounds for the prevention and treatment of adhesions, and to develop pharmaceutical compositions containing a sufficient amount of one and/or several of the compounds described above and a pharmaceutically acceptable carrier, diluent or excipient.

Another purpose of the invention is to develop a method for adhesion prevention allowing to avoid additional administration of medications in the postoperative period.

The inventors of the present invention found out that p38 MAP-kinase inhibitors may be used for the treatment and prevention of adhesion formation. In particular, a compound [4-(4-fluorophenyl)-2-(4-methylsulfonyl-phenyl)-5-(4-pyridyl)-lH-imidazole], also known as SB203580 (chemical formula is shown in A.Cuenda et. al, FEBS Letters 364(1995) 229-233), and other new compounds of type (I)-(VII) (described below), prepared according to the present invention at the concentration of 0.1 to 100 pg/ml (on the basis of active substance inhibiting p38 MAP-kinase activity) and in the amount of 0.1 to 500 ml (depending on serous sac surface), sufficient to moisten the serous sac surface, provide a barrier to adhesion formation in the injured serous sac (Fig.l).

The present invention is characterised by the development of new compounds for the prevention and treatment of adhesions showing an inhibiting activity targeted at excessive proliferation response when involved in pathological processes of serous surfaces, and by the development of pharmaceutical compositions containing a sufficient amount of one and/or several compounds for adhesion prevention, together with a method for the prevention and treatment of adhesions using these compounds.

The group of the compounds described above may be characterised by the following structural formulas.

Type (I):

or type (II):

or type (III):

or type (IV):

or type (V):

or type (VI):

or type (VII):

where Ri is a base unit of water soluble polymers of natural or synthetic origin; x , y and z — integrer values, where x, y and z φ 0. X, Y and Z values depend on the number of monomer units in a polymer molecule. In fact, each molecule has n of monomers. Some of these monomers bind to the p38 MAP-kinase inhibitor, the rest of the molecule remains unbound. Thus, (X+Y) = n (or in some cases X+Y+Z = n, and n<6000), where n may be any integer values. In one embodiment of the present invention, x+y+z >10.

The present invention relates to the use of SB203580 for new indications, namely as a compound exhibiting anti-adhesion activity.

Moreover, the present invention relates to pharmaceutical compositions that are characterised by a sufficient amount of any of the described compounds of type (1)-(VII), or their combinations, or compound SB203580, or its combinations with any of the compounds of type (I)-(VII), and a pharmaceutically acceptable carrier, diluent or excipient.

The amount of the active ingredient in the pharmaceutical composition, namely the amount of the compound of type (I)-(VII) or compound SB203580 or their combinations, sufficient to achieve therapeutic effects, may vary depending both on the compound used or the route of its administration, and on the area of serous sac surface in a treated patient.

The acceptable dose of the compound of type (I)-(VII) or compound SB203580 used for the treatment of serous sac surface is about 0.01 pg to 50 mg on the basis of a compound inhibiting p38 MAP-kinase activity.

Although the active ingredient may be administered separately as a raw chemical substance, it is preferable to include it into the pharmaceutical composition. The amount of the active ingredient is also preferable to be 0.00001% to 99.99999% of the total pharmaceutical composition volume.

Therefore, drug formulations may be presented in the form of standard dosage units or single doses and may be prepared using any of the known pharmaceutical methods. Methods described in A.I. Tikhonov, T.G. Yarnykh “Medication Technology”, published by NPU 2002, pages 228, 229, 242, may be used as one of the alternatives to prepare the pharmaceutical composition. All methods include the phase of interaction the active ingredient to the carrier, which consists of one or more excipients. Pharmaceutical compositions are usually prepared by the steady and close contact (of the active ingredient with the liquid carrier.

The pharmaceutical composition according to the present invention can be prepared and administered in liquid form for perfusion systems, in the form of spray, spraying and vaporisation solution, foamy aerosol, gel or suspension, or in any other liquid form.

As regards to the route of administration, it is appropriate to apply the solution over the serous sac surface, including wounds and organs, or to spray the solution for the prevention of adhesions using a special sprayer immediately after its preparation.

Once prepared, the adhesion prevention solution can be sprayed over the necessary areas, and the solution used for adhesion prevention in the wound areas also can be sprayed evenly over the necessary areas. The areas of potential adhesion formation can also be thoroughly sprayed.

For spraying the solution, a sprayer with two pressure pulverisers can be used, in which drops of the solution are transferred by air or carbon dioxide, or a sprayer with one pressure pulveriser, in which the solution turns into small particles.

The technical result of the invention lies in the fact that the compounds of type (I)-(VII) are generated by conjugation of the base polymer and a protonated derivate of pyridine-imidazole or pyridine-pyrrole, and that the pharmaceutical composition containing a sufficient amount of the compound of type (I)-(VII) and/or compound SB203580, and a pharmaceutically acceptable carrier, diluent or excipient has been prepared.

Any appropriate base polymer can be used to generate compounds of type (I)-(VII). It is preferable to use as base polymers polyethylenimine and its copolymers, polyvinylpyridines and their copolymers, polyvinylimidazole and its copolymers, polyvinyltriazole and its copolymers, chitosan and its derivates, carboxymethyl cellulose salts, polyacrylic acid and its copolymers, polymethacrylic acid and its copolymers, or polymethylmethacrylic acid and its copolymers.

The invention also requires that the pharmaceutical composition is administered intraperitoneally during surgical, minimally invasive or diagnostic procedures for the prevention or treatment of any disease or medical condition associated with adhesion formation and/or development.

The method for prevention of adhesions consists in the following: An appropriate p38 MAP-kinase inhibitor is injected into the serous sac immediately after operative and/or diagnostic procedures.

As one of the variant for adhesion prevention, p3 8 MAP-kinase inhibitors, in particular SB203580 or one of the compounds of type (I)-(VII) or their combinations are injected in the sterile solution form at the concentration of 0.1 to 100 pg/ml (on the basis of active substance inhibiting p38 MAP-kinase activity) and in the sufficient amount to moisten the serous sac surface. The solution is administered once at a dose that may inhibit not less than 50% of p38 MAP-kinase activity in injured areas. A specific feature of the method is that with a disease or medical condition associated with serous sac disorders the pharmaceutical composition containing SB203580 or one of the compounds of type (I)-(VII) or their combination is administered by the surgical subject.

The method can be used for the treatment of diseases or medical conditions accompanied by exudation or bleeding in the serous sac, or by damage to the serous membrane.

Compounds intended for adhesion prevention, pharmaceutical compositions containing these compounds and the method for the prevention and treatment of adhesions described in the present invention may be used in the fields of experimental and clinical medicine, and/or veterinary practice. Its functionality has been confirmed by the distinctive characteristics and features described above.

Thus, the inventors have demonstrated convincing evidence that p38 MAP-kinase inhibitors may be used as medications with anti-adhesion activity.

Moreover, the inventors have achieved the aim of generating compounds that are effective in the treatment and/or prevention of adhesions, and developed pharmaceutical compositions using these compounds with a sufficient amount of one of the compounds and/or their combination and a pharmaceutically acceptable carrier, diluent or excipient. Moreover, the inventors have developed an effective method for adhesion prevention allowing to avoid additional administration of medications in the postoperative period.

BRIEF DESCRIPTION OF DRAWINGS

Drawing 1 (therapeutic efficacy of compound SB203580, a p38 MAP-kinase inhibitor)

Drawing 1 shows a histological section of the intestinal wall in the area of adhesion formation. No signs of adhesions are shown in the animal from the experimental group, even at the abdominal trauma zone and in the area of the postoperative suture (position C), van Gieson's staining.

Drawing 2 (therapeutic efficacy of compound SB203580, a p38 MAP-kinase inhibitor)

Drawing 2 shows a histological section of the intestinal wall in the area of adhesion formation in an animal from the control group (van Gieson's staining). The observed adhesions are characterized by a greater length, density of connective tissue (position A), and signs of vascularisation (position B).

Drawing 3 (therapeutic efficacy of p38 MAP-kinase inhibitor — compound of type (I), where R1 is a base unit of polyvinylimidazole)

Drawing 3 shows a histological section of the intestinal wall in the area of adhesion formation (position A) in an animal from the control group 30 days after modelling (van Gieson's staining). Intestinal wall adhesions (position A) and well-vascularised adhesions (position B) are shown.

Drawing 4 (therapeutic efficacy of p38 MAP-kinase inhibitor — compound of type (I), where R1 is a base unit of polyvinylimidazole)

Drawing 4 shows a histological section of the intestinal wall in the area of adhesion formation. No signs of adhesions in the animal from the experimental group are evident, even at the abdominal trauma zone and in the area of the postoperative suture (position C), 30 days after modelling (van Gieson's staining).

Drawing 5 (therapeutic efficacy of p38 MAP-kinase inhibitor — compound of type (I), where R1 is a base unit of polyvinylimidazole)

Drawing 5 shows the severity of adhesion formation on Day 7, 14 and 28 in the control group compared to that in the experimental group.

Drawing 6 UV-VIS spectrum of protonated salt aqueous solution of compound of type (I), where R1 is a base unit of polyvinylimidazole. Line 1 - water, Line 2 -aqueous solution of polyvinylimidazole, Line 3 - compound (I), Line 4 - compound (I)+polymer polyvinylimidazole.

Drawing 7 UV-VIS spectrum of protonated salt aqueous solution of compound of type (I), where R1 is a base unit of carboxymethyl cellulose. Line 1 - water, Line 2 -aqueous solution of polyvinylimidazole, Line 3 - compound (I), Line 4 - compound (I)+polymer carboxymethyl cellulose.

Drawing 8 UV-VIS spectrum of protonated salt aqueous solution of compound of type (I), where R1 is a base unit of polyvinyltriazole. Line 1 - water, Line 2 - aqueous solution of polyvinylimidazole, Line 3 - compound (I), Line 4 - compound (I)+polymer polyvinyltriazole.

DETAILED DESCRIPTION OF PREFERRED IMPLEMENTATION VARIANTS OF THE INVENTION

The following examples are used as illustration, but not to limit the scope of the present invention.

Example 1

Preparation of the compounds of type (I)-(VII) with anti-adhesion activity

Compounds of type (I)-(VII) are prepared during the three-phase process according to schemes 1-3 given below.

During the first phase, an aqueous-based polymer solution, for example, polyvinylimidazole, is prepared according to scheme 1 for the synthesis of the compounds of type (I)-(VII) with anti-adhesion activity.

During the second phase, aqueous protonated solutions of the compounds of type (I)-(VII) are prepared by dissolving them in the aqueous solution of any nonorganic or organic acid (HAn) according to scheme 2. Record UV-VIS spectra of aqueous solutions of the compounds of type (I)-(VII).

During the third phase, mix the obtained aqueous polymer solution with the aqueous solution of the compounds of type (I)-(VII), leave for 1 hour at room temperature until complete degradation of protonated salts (degradation of salts exposed to the high-alkaline medium of aqueous polymer solutions) forming the compound of type (I)-(VII) as a conjugate of the base polymer and active ingredient (scheme 3).

Record UV-VIS spectra again and according to their transformation (compared to protonated salts), state a formation of the compound of type (I)-(VII) with polymers.

These compounds of type (I)-(VII), as opposed to parent compounds without polymers, may be used for the treatment and prevention of adhesion formation.

Scheme 1.

Preparation of aqueous polymer solution

Preparation of aqueous polyvinylimidazole solution

Preparation of aqueous carboxymethyl cellulose solution

R=H,CH2C(0)0M; M=Na+, K+, NH4+ etc.

Preparation of aqueous chitosan solution

Scheme 2

Preparation of water-soluble protonated compounds of type (I)-(VII)

Scheme 3

Preparation of the compounds of type (I)-(VII)

Example 2

According to schemes 1-3 given in example 1 (see above), a compound of type (I) was generated on the basis of chitosan, a polymer of natural origin:

, where x and y — integral values, x, y ^0.

Example 3

According to schemes 1-3 given in example 1 (see above), a compound of type (I) was generated on the basis of polyvinylimidazole, a polymer of synthetic origin:

, where x and y — integral values, x, y Φ 0.

Example 4

According to schemes 1-3 given in example 1 (see above), a compound of type (I) was generated on the basis of carboxymethyl cellulose, a polymer of synthetic origin:

, where x and y — integral values, x, y Φ 0.

Example 5

Adhesions in the peritoneal cavity were generated in laboratory animals (Wistar rats, 9 months of age, weight of 220-250 g) by injuring the surface of caecum and abdominal wall scarification in the injured area. The study was conducted in compliance with the principles of the European Convention for the

Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, France, 1986), as well as with regulations for humane treatment specified in Guidance for Proper Conduct of Animal Experiments (Attachment to Order No. 755 of the Ministry of Health of the USSR, dated August 12, 1977).

Ten laboratory animals were used in this study. They were divided into 2 groups: the experimental group and the control group.

Before celiorrhaphy, 3 ml of sterile solution SB203580, p38 MAP-kinase inhibitor, at the concentration 10 pg/ml was injected once into the experimental animals. The calculation was based on the minimum volume of solution required to moisten the peritoneal surface (diZerega G.S. Peritoneum, peritoneal healing and adhesion formation/ G.S. diZerega// in: Perotoneal surgery: Ed. G.S. diZerega.- Berlin-Heidelberg-New York: Springer, 2006.- P. 3-38), and the concentration was calculated on the basis of IC50 in the boundary layer of cells. The control animals received a corresponding amount of normal saline solution.

On Day 28 after the abdominal cavity being injured, all animals were autopsied for the examination of abdominal cavity organs and the assessment of the severity and incidence of adhesions, deformation of abdominal cavity organs and distribution structures of different types of adhesions. The severity of adhesions was scored according to micro- and macroscopic adhesion scales [Micronized purified flavonoid fraction may prevent formation of intraperitoneal adhesions in rats// H.G. Yilmaz, I.H. Tacyildiz, C. Keles et al.// Fertil. Steril.-2005.- Vol. 84, Suppl. 2.- P 1083-1087],

The visceral peritoneum and abdominal organs involved in adhesion formation were examined histologically after fixation in FineFIX solution (Milestone), paraffin filling, staining with hematoxylin-eosin and van Gieson's staining.

Adhesions in the peritoneal cavity were observed in 100% of control animals, and intestinal wall adhesions were detected in 100% of cases. Thus, Fig. 2 shows a histological section of the intestinal wall in the area of adhesion formation (position A) in the animal from the control group (van Gieson's staining). The observed adhesions were characterized by a greater length and density of connective tissue, and signs of vascularisation (position B).

No cases of intestinal wall adhesions were observed in the experimental animals. Fig. 1 (Attachment to Application) shows no signs of adhesions in the animal from the experimental group, even at the abdominal trauma zone and in the area of the postoperative suture (position C), van Gieson's staining.

The severity of adhesion formation was scored as 7 in the control group, and as 2 in the experimental group (p<0.01).

Results of the study suggest that the described method may be used for the prevention of adhesions in the serous sac after surgical interventions.

Thus, the described method is considered to be efficient in preventing adhesion formation when the specified medication is injected once immediately after the operative procedures, and results in minimal injuries and simplifies the prevention of adhesions, while it decreases the risk of organ injuries and the risk of infections in the serous sac.

Example 6

Adhesions in the peritoneal cavity were generated in laboratory animals (Wistar rats, 9 months of age, weight of 220-250 g) by injuring the surface of caecum and abdominal wall scarification in the injured area. The study was conducted in compliance with the principles of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, France, 1986), as well as with regulations for humane treatment specified in Guidance for Proper Conduct of Animal Experiments (Attachment to Order No. 755 of the Ministry of Health of the USSR, dated August 12, 1977).

Thirty laboratory animals were used in this study. They were divided into 2 groups: the experimental group and the control group.

Before celiorrhaphy, a pharmaceutical composition (3 ml) containing the compound according to example 2, at the concentration 2 □ 10' mol/1 was injected once in experimental animals. The calculation was based on the minimum volume of solution required to moisten the peritoneal surface [diZerega G.S. Peritoneum, peritoneal healing and adhesion formation/ G.S. diZerega// in: Perotoneal surgery: Ed. G.S. diZerega.- Berlin-Heidelberg-New York: Springer, 2006.- P. 3-38]). The control animals were injected with a corresponding amount of saline solution.

On Day 7, 14 and 28 after the abdominal cavity being injured, all animals were autopsied for the examination of abdominal cavity organs and the assessment of the severity and incidence of adhesions, deformation of abdominal cavity organs and distribution structures of different types of adhesions. The visceral peritoneum and abdominal organs involved in adhesion formation were examined histologically after fixation in FineFIX solution (Milestone), paraffin filling, staining with hematoxylin-eosin and van Gieson's staining.

Adhesions in the peritoneal cavity were observed in 100% of the control animals, and intestinal wall adhesions were detected in 100% of cases. The observed adhesions were characterised by a greater length and density of connective tissue, and signs of vascularisation (Attachment to Application, Fig. 3). Also, Fig. 3 shows a histological section of the intestinal wall in the area of adhesion formation (position A) in the animal from the control group 30 days after modelling (van Gieson's staining). Intestinal wall adhesions (position A) and well-vascularised adhesions (position B) were shown.

No cases of intestinal wall adhesions were observed in experimental animals. Fig. 4 shows no signs of adhesions in the animal from the experimental group, even at the abdominal trauma zone and in the area of the postoperative suture (position C), 30 days after modelling (van Gieson's staining).

On Day 7, 14 and 28, the severity of adhesion formation was significantly higher in the control group than in the experimental group (Figure 5).

Example 7

Adhesions in the peritoneal cavity were generated in laboratory animals divided into 8 experimental groups, each composed of 25 individuals (Wistar male rats, 9 months of age, weight of 180-200 g) to evaluate the effects of the compounds of type (I)-(VII) on the basis of chitosan, carboxymethyl cellulose, polyvinyltetrazole, polyethylenimine, polyvinyltriazole, and polyacrylic acid on the prevention and course of adhesions. The study was conducted according to the methods described in example 5 and 6 of the present invention.

Compounds of type (I)-(VII) on the basis of chitosan, carboxymethyl cellulose, polyvinyltetrazole, polyethylenimine, polyvinyltriazole, and polyacrylic acid (at a dose of 10 mg/kg based on the compound inhibiting p38 MAP-kinase activity) were injected in the sterile solution form in animals of all experimental groups (once, after the surgical intervention) after the modelling of adhesion formation.

The control animals were injected with a corresponding amount of saline solution.

Adhesions in the peritoneal cavity were observed in 100% of the control animals, and intestinal wall adhesions were detected in 100% of cases.

No cases of intestinal wall adhesions were observed in any of 6 experimental groups.

On Day 7, 14 and 30, the severity of adhesion formation was significantly higher in the control group than in the experimental group.

Example 8

Preparation of conjugates of polyvinylimidazole and 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-lH-imidazole (compound of type (1))

Water solution of polyvinyl imidazole is prepared according to abovementioned scheme 1 on page 20 of the description.

Then on the second stage water solutions of protonated salt of type (I) compound are prepared by its dissolution in water solution of hydrochloric acid according to scheme 2 on page 21 of the description. UV-VIS spectra of acquired water solution of protonated salt of type (I) compound are registered.

On the third stage according to scheme 3 on page 22 of the description acquired water solution of polyvinylimidazole and water solution of protonated salt of type (I) compound are mixed, held for 1 hour at room temperature for complete reverse decay of protonated salt of compound with formation of water solution of conjugate of polyvinylimidazole and salt of 4-[4-(4-fluoropheny 1)-2-(4-methylsulphinylphenyl)-1 H-imidazolyl-5-pyridine (hereafter “conjugate”). UV-VIS spectra are registered again and by its transformation (in comparison with protonated salt) formation of compound of type (I) with polyvinyl imidazole is registered. lH 600 MHz: (5% weight B H20-d2, 25°C, JEOL ECA 600, ppm): 1,8-2,2 (2H, CH2 group in polymer), 2.7 (3H, CH3 group in imidazole compound), 2.4-2.8, 3.0-3.2 n 4.6-4.7 (1H, CH group in syndiotactic and geterotactic fragments of polymer), 6.4-7.1 (3H, CH groups of imidazole ring in polymer), 7,3, 7,4, 7,5, 7,7, 8,0, 8,3 (16H, CH groups of aromatic rings in imidazole compound).

Example 9

Preparation of conjugate of polymethacrylate 4-(2-(4-fluorophenyl)-5-(4-methylsulphinyl)phenyl)-lH-pyrrole-3-yl)pyridine (compound of type (VII))

The conjugate is prepared according to schemes 1-3 on pages 20-22 of the description.

Conjugate efficiency was 96%.

At room temperature *H NMR spectrum was registered at ECA 600spectrometer with working frequency for nuclei 1H 600 MHz: (5% weight B H20-d2, 25°C, JEOL ECA 600, ppm): 1.5 (3H, signal CH3 in polymer), 1.9-2.3 (2H, two signals CH2 in polymer), 2.7 (3H, CH3 group of pyrrole compound), 7.0, 7.3, 7.4, 7.5, 7.7, 8.0, 8.3, (16H, CH groups of aromatic rings).

Example 10

Preparation of conjugate 4-(4-(4-fluorophenyl)-2-(4-(methylsulphinyl)phenyl)-lH-imidazolyl)pyridine with polyvinyl( 1 H-imidazole)4-(4-(4-fluoropheny 1)-2-(4- methylsulphinyl)phenyl)-lH-imidazolyl)pyridine (compound of type (III)).

Conjugate efficiency was 97%.

At room temperature Ή NMR spectrum was registered on ECA spectrometer with working frequency for nuclei 'H 600 MHz: (5% weight B H20-d2, 25°C, JEOL ECA 600, ppm): 1,9-2,3 (2H, CH2 group in polymer), 2.7 (3H, singlet of CH3 group in imidazole compound), 2.5-3.2 n 4.6-4.7 (1H, CH group in syndiotactic and geterotactic fragments of polymer), 6.6-7.2 (3H, CH groups of imidazole ring in polymer), 7,3-7,6, 8,1, 8,3 (16H, CH CH groups of aromatic rings in imidazole compound).

Example 11

Preparation of conjugate of polyvinyltetrazole and 4-[5-(4-iodo-phenyl)-2-(4-methylsulphinylphenyl)-3H-imidazole-4-yl]-pyridine (compound of type (IV))

Conjugate efficiency was 95%.

At room temperature IR spectrum in table KBr was registered on spectrometer Perkin-Elmer 1310, cm'1: 3300(N-H, C-H oscillations); 2200-1600(peculiarities of oscillations C=C, C=N in N-heteroaromatic polymers); 15001400 (aromatic C=C oscillations); 1000-900 (=C-H, S=0).

Example 12

Preparation of conjugate of chitosan and 4-(5-(3-iodophenyl)-2-(4-(methylsulphinyl)phenyl)-lH-pyrrolyl-4-yl)pyridine (compound of type (VII))

Conjugate efficiency was 96%.

At room temperature Ή NMR spectrum was registered on JEOL ECA 600 spectrometer with working frequency for nuclei 'H 600 MHz: (5% weight B H20-d2, 25°C, JEOL ECA 600, ppm): 2.7 (3H, signal CH3 of imidazole compound), 3.1-3.3 (1H, CH group of chitosan), 3.6-4.1 (4H, CH groups of chitosan), 4.9-5.0 (1H, CH group of chitosan), 7.2-7.5 (6H, CH groups of phenyl rings of imidazole compound), 7.8, 8.1, 8.3 (6H, CH groups of phenyl rings of imidazole compound).

Example 13

During conduction of scientific studies of commissural formation process in abdomen we have discovered that intensity of commissural process correlates to activity of p38 MAPK in submesothelial layer of cells in 3-7 days after procedures on organs, covered with serous membrane. During artificial decrease of p38 MAPK activity in cells of subserous tissues it was revealed that commissural formation either stops or significantly decreases, and process of mesothelization, based on homing of mesotheliocytes in focuses of deserousing, does not undergo significant changes. These molecular biological studies, first in the world, allowed to develop methods of preventive care of commissural formation.

Authors of the study examined content of phosphorylated (active) form of p38-phospho MAPK by culture of fibroblasts. 5 groups were studied and in cultural medium of three groups at the beginning of cultivation one of the claimed substances was added once: group 1 - substance by formula III, in which polyvinyltetrazole is used as a polymer, and values of x, y and z, received at NMR study, were 18, 5 and 1, respectively; group 2 - substance by formula VI, in which sodium salt of carboxymethylcellulose was a polymer, and ratio of coefficients x, y, z was 16:7:2, group 3 - substance by formula VII, in which chitosan was a polymer and x:y ratio was 8:1. In group 4 SB203580 was added in cultural medium, and in control group the medium was used without any additions.

Activity of p38-phospho MAPK was determined in lysates of cells in 7 days of incubation using set Phospho p38 (Τ180/Ύ182) Flex Set (BD Bioscience) on running cytometer FACScalibur.

Prior to determination of content of phosphorylated form of p38 MAPK the culture was stimulated by addition of mitogen - FGF2.

Content of active form of p38-phospho MAPK is presented on the diagram:

High value of p38phospho activity in group SB203580 caught our attention,

which was unexpected. However, introduction of the substance in combination with FGF2 led to expected abrupt decrease of content of active form p38 MAPK to 1038±215 U/ml, that allows state short effect of the substance and its absence in 7 days of incubation whereas conjugates with polymers continue to demonstrate their activity.

Cell culture of lung fibroblasts of Wistar rats was prepared by tissue homogenization, its treatment with enzymes for cell separation and inoculation in nutritive medium with addition of embryonic serum. After that zero inoculation was incubated in C02 incubator, and grown colonies of fibroblasts were reseed for further studies. Commercially available z38 MAP kinase inhibitors were used, VX-702 and TAK-715 inhibitors, in particular, that were acquired from AbcamBiochemicals, and SB239063 inhibitor from Tocris Bioscience. The inhibitors were added to nutritive medium during changing.

Further during short incubation (6 hours) on primary culture of lung fibroblasts of Wistar rats a possibility of p38 MARK activity by substances consistent with claimed compounds by formulas (I)-(VII) was studied, in which p38 MAP kinase inhibitor conjugated with polyvinyltetrazole, and also separately with compounds: 1. trans-4-[4-(4-fluorophenyl)5-(2-methoxy-4-pyrimidinyl)-lH-imidazole-l-yl]cyclohexanol (SB239063); 2. 6-(N-carbamoyl-2,6-difluoroanilino)-2-(2,4-difluorophenyl)pyridine-3-carboxamide (VX-702); 3. N-[4-[2-ethyl-4-(3-methylphenyl)-l,3-thiazole-5-yl]-2-pyridyl]benzamide (TAK-715), that are selective inhibitors of p38 MAPK and have significant differences in structure than non-polymeric parts of composites by formulas (I)-(VII).

As a result of the study it was determined that all studied substances have an inhibiting effect on p38 to a greater or lesser extent preventing from t formation of its active form. Activity of p38 phospho in comparison with control (addition of placebo, designated as 100%) is presented on the following Figure.

Example 14

Preparation of conjugate of polyacrylate and 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-lH-imidazole (compound of type (1))

The conjugate is prepared according to example 1. Conjugate efficiency was 97,8%.

At room temperature !H NMR spectrum was registered at ECA 600 spectrometer with working frequency for nuclei 'H 600 MHz: (5% weight B H20-d2, 25°C, JEOL ECA 600, ppm): 1.9-2.1 (2H, CH2 group in polymer), 2.3-2.6, 3.03.1 and 4.1-4.3 (1H, signals in syndiotactic, heterotactic and isotactic fragments of CH group in polymer), 2.7 (3H, singlet of CH3 group of imidazole compound), 7.3, 7.4, 7.5, 7.7, 8.0, 8.3 (by 2H, CH groups of phenyl rings of imidazole compound).

Example 15

Preparation of conjugate of polyethyleneimine and 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-lH-imidazole (compound of type (1))

The conjugate is prepared according to example 1. Conjugate efficiency was 99,2%.

At room temperature Ή NMR spectrum was registered at ECA 600 spectrometer with working frequency for nuclei 'H 600 MHz: (5% weight B H20-d2, 25°C, JEOL ECA 600, ppm): 1.4-1.5 (2H, CH2 group in polymer), 1.8-1.9 (2H, CH2 group in polymer), 2.6 (3H, singlet of CH3 group of imidazole compound), 7.0, 7.2, 7.3, 7.4, 7.7, 7.9 (by 2H, CH groups of phenyl rings of imidazole compound).

Claims

Claims

1. Use of compounds p38 MAP kinase inhibitors in the manufacture of a medicament for preventing the formation and/or growth of adhesions of serous cavity.
2. Use according to claim 1, wherein p38 MAP kinase inhibitor is a compound 4-(4-fluorinephenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-lH-imidazole.
3. Use according to claim 1, wherein the compound of p38 MAP kinase inhibitor is a compound of type (I): or (II):

or (III):

or (IV):

or (V):

or (VI):

or (VII):

where Ri - structural unit of water-solvable polymers of basic character of natural or synthetic origin including salts of polyacrylic acid, polymethacrylic acid, polymethylmethacrylic acid and/or their copolymers and derivatives, polyethylenimine and/or its copolymers, polyvinylpyridines and/or their copolymers, polyvinylimidazole and/or its copolymers, polyvinyltriazole and/or its copolymers, polyvinyltetrazole and/or its copolymers, chitosan and/or its derivates, carboxymethyl cellulose; An — acid residual; x, y and z are integer values, besides if x>l, y>l, z=0, than x+y=n, if x>l, y>l, z>l, than x+y+z=n, wherein n - the number of monomer units in a polymer molecule, wherein n is integral number and n<6000.
4. Use according to claim 3, wherein R1 is structural units of chitosan and/or its derivatives.
5. Use according to claim 3, wherein R1 is structural units of polyvinyltetrazole and/or its copolymers.
6. Use according to claim 3, wherein R1 is structural units of carboxymethylcellulose salts.
7. Use according to claim 3, wherein R1 is structural units of polyvinyl imidazole and/or its copolymers.
8. Use according to claim 3, wherein R1 is structural units of polyethylenimine and/or its copolymers.
9. Use according to claim 3, wherein R1 is structural units of polyvinylpyridines and/or their copolymers.
10. Use according to claim 3, wherein R1 is structural units of polyvinyltriazoles and/or their copolymers.
11. Use according to claim 3, wherein R1 is structural units of polyacrylic acid salts and/or its copolymers.
12. Use according to claim 3, wherein R1 is structural units of polymethacrylic acid salts and/or its copolymers.
13. Use according to one of claims 3-12, wherein p38 MAP kinase inhibitor is any of type (I)-(VII) compounds or its combination with each other or with compound 4-(4-fhrorinephenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)-1H-imidazole.
14. Compound p38 of MAP kinase inhibitor of type

or (II):

or (III):

or (IV):

or (V):

or (VI):

or (VII):

where R1 is a base unit of water soluble polymers of natural or synthetic origin including salts of polyacrylic acid, polymethacrylic acid, polymethylmethacrylic acid and/or their copolymers and derivatives, polyethylenimine and/or its copolymers, polyvinylpyridines and/or their copolymers, polyvinylimidazole and/or its copolymers, polyvinyltriazole and/or its copolymers, polyvinyltetrazole and/or its copolymers, chitosan and/or its derivates, carboxymethyl cellulose; An — acid residual; x, y and z are integer values, besides if x>l, y>l, z=0, than x+y=n, if x>l, y>l, z>l, than x+y+z=n, wherein n - the number of monomer units in a polymer molecule, wherein n is integral number and n<6000.
15. A compound according to claim 14, where R1 is a base unit of chitosan and its derivates.
16. A compound according to claim 14, where R1 is a base unit of polyvinyltetrazole and/or its copolymers.
17. A compound according to claim 14, where R1 is a base unit of carboxymethyl cellulose salts.
18. A compound according to claim 14, where R1 is a base unit of polyvinylimidazole and/or its copolymers.
19. A compound according to claim 14, where R1 is a base unit of polyethylenimine and/or its copolymers.
20. A compound according to claim 14, where R1 is a base unit of polyvinylpyridines and/or their copolymers.
21. A compound according to claim 14, where R1 is a base unit of polyvinyltriazole and/or its copolymers.
22. A compound according to claim 14, where R1 is a base unit of polyacrylic acid salts and/or its copolymers.
23. A compound according to claim 14, where R1 is a base unit of polymethacrylic acid salts and/or its copolymers.
24. A pharmaceutical composition containing a sufficient amount of any of compounds according to claims 14-23 or its combinations, and a pharmaceutically acceptable carrier, diluent or excipient.